The actin binding and ATPase kinetics of cardiac myosin subfragment-1 were compared with prior studies on skeletal myosin subfragments. Previous kinetic studies on rabbit skeletal subfragment-1 (S-1) have revealed two important features of the actomyosin ATPase activity. First, hydrolysis of ATP by myosin subfragment-1 proceeds both when S-1 is bound to actin and when it is dissociated from actin. Second, the actin concentration required to reach half the maximum ATPase activity, Ka(ATPase), is considerably lower than the actin concentration required to bind half the subfragment-1 during steady state hydrolysis of ATP, Ka(binding). These kinetic facts require that skeletal myosin hydrolyze ATP without dissociating from actin; therefore, a "nondissociating" pathway for ATP hydrolysis exists. The studies reported here show that porcine cardiac S-1 is very similar to rabbit skeletal S-1. Under identical conditions to prior work on skeletal S-1, the Ka(ATPase) of porcine cardiac S-1 is approximately equal to that reported for skeletal S-1. This is also true for Ka(binding). Comparison of Ka(ATPase) and Ka(binding) shows that for cardiac proteins Ka(ATPase) is fourfold to sixfold stronger than Ka(binding), i.e., half maximal ATPase activity is achieved at about one fifth the actin necessary to reach 50% binding. The extrapolated maximum ATPase activity at saturating actin concentration for cardiac S-1 is consistently slower than skeletal S-1 by about a factor of 2.5. Furthermore, studies of the actoS-1 ATPase activity at high actin concentrations as well as with crosslinked actoS-1 show no significant inhibition, implying the requirement of a "nondissociating" pathway for ATP hydrolysis by cardiac myosin subfragment-1.

The actin dependence of the rate and magnitude of the initial phosphate burst was measured using both quench-How and stopped-flow kinetic techniques. These studies revealed that even at high actin concentrations the magnitude of the phosphate burst was a significant fraction of the magnitude that exists in the absence of actin. Furthermore, it was shown that the rate of the burst rises rapidly as a function of the actin concentration. Detailed modeling with the four-state model revealed that if the predicted V^ is constrained to be approximately equal to the extrapolated value (double reciprocal plot) and if the apparent dissociation constant of subfragment-1 to actin divided by the apparent activation constant of the actin-activated myosin ATPase activity (K^^^/K ArPmK ) is constrained to be considerably different from one, then the model is unable to simultaneously account for the ATPase activity and the rate and magnitude of the initial inorganic phosphate burst. (Circulation Research 1989;65:515-525) R ecent studies on the steady-state properties of porcine cardiac subfragment-1 (S-l) 1 have revealed that the biochemical kinetics of cardiac S-l are very similar to the kinetics of rabbit skeletal S-l.2 It was shown that ^bindmgj the apparent dissociation constant of S-l to actin, and /CATP»K> the apparent activation constant of the actin-activated myosin ATPase activity, of porcine cardiac S-l were, within experimental error, equal to those of skeletal S-l and that the ratio of these constants (i.e., Ktinding/iCATp^) for the cardiac proteins is approximately 5 : 1. The only significant difference between skeletal and cardiac S-l was that the extrapolated V^, of the double reciprocal plot 3 was approximately 2/sec for cardiac S-l and 4-5/sec for skeletal S-l at 15° C and low ionic strength (0.013 M). Furthermore, it was shown that cross-linked actoS-1, prepared using the zero-length cross-linking agent l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), possesses a significant ATPase activity. 4 ' 5 This lack of significant inhibition of the ATPase activity of cardiac actoS-1 at saturating actin con- centrations was reported previously for skeletal S-l, and detailed kinetic modeling revealed that a "nondissociating pathway" for ATP hydrolysis was required to account for the data. 5 It was then concluded that the four-state model was the minimal model that could account for the data (see Figure I).1 Because the steady-state kinetics of cardiac S-l are almost identical to rabbit skeletal S-l, except for a significantly different V^, it becomes interesting to pursue the kinetic mechanism of this difference. The important question is whether a four-state model can adequately account for the cardiac data or whether a more complex model (e.g., a six-state model) needs to be postulated. In the current work, we have expanded our prior kinetic studies of cardiac S-l to include presteady-state measurements of the rate and magnitude of the initial phosphate burst, both in the presence and absence of actin. As wa...

We have investigated the effect of limited trypsin digestion of chymotryptic myosin Subfragment-1 (S-1) on its kinetic properties. We find that Vmax (i.e., the extrapolated maximal ATPase activity at infinite actin) remains approximately constant, independent of the period of digestion. We also find that the apparent actin activation constant, KATPase, and the apparent dissociation constant, Kbinding, are both significantly weakened by trypsin digestion of S-1, and that these kinetic parameters change in concert. In addition, we investigated the effect of limited trypsin digestion on the initial phosphate burst. We find that trypsin digestion has no effect on the rate of the tryptophan fluorescence enhancement that occurs after ATP binds to digested S-1, but that the magnitude of the fluorescence enhancement falls approximately 40% with digestion. Digested S-1 also showed anomalous behavior in that the fluorescence magnitude increased and the fluorescence rate dropped in the presence of actin. Trypsin digestion also decreased the magnitude of the chemically measured Pi burst approximately 35%, but this magnitude was essentially unaffected by actin. A possible explanation for this behavior is discussed.

The 10S-->6S (Flexed-->Extended) transition in smooth muscle myosin is related to increased ATPase activity, but there is controversy over whether the analogous 9S-->7S transition in HMM is also associated with ATPase activity. We therefore studied the association of ionic strength, phosphorylation, and ATPase activity for HMM as compared to S1 which has no apparent flexed conformation. In addition, we performed both steady state and single turnover analyses, to control for artifacts due to multiple subfragment populations that might skew steady state results. At low ionic strength where myosin and HMM are in the flexed conformation, HMM had a near zero ATPase activity while S-1 had a high ATPase rate (0.07 s-1). At 400 mM ionic strength, where both myosin and HMM are in the extended conformation, S1 and HMM had the same ATPase rate (0.04 s-1). Phosphorylation did not affect S1 significantly, but shifted the HMM curve to higher rates at lower ionic strengths. Both steady state and single turnover experiments gave the same results, indicating that steady state results were not skewed by multiple subfragment populations. These data indicate that HMM has a conformation-ATPase relation similar to that observed with myosin. Furthermore, these findings suggest that the S1 ATPase rate corresponds to that of HMM in the extended conformation.